Smchd1 regulates long-range chromatin interactions on the inactive X chromosome and at Hox clusters.

The regulation of higher-order chromatin structure is complex and dynamic, and a full understanding of the suite of mechanisms governing this architecture is lacking. Here, we reveal the noncanonical SMC protein Smchd1 to be a novel regulator of long-range chromatin interactions in mice, and we add Smchd1 to the canon of epigenetic proteins required for Hox-gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes that depend on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we found increased short-range interactions and ectopic enhancer activation. In contrast, the inactive X chromosome was transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. In the inactive X, we observed spreading of trimethylated histone H3 K27 (H3K27me3) domains into regions not normally decorated by this mark. Together, these data suggest that Smchd1 is able to insulate chromatin, thereby limiting access to other chromatin-modifying proteins.

Smchd1 regulates a subset of autosomal genes subject to monoallelic expression in addition to being critical for X inactivation.

BACKGROUND: Smchd1 is an epigenetic modifier essential for X chromosome inactivation: female embryos lacking Smchd1 fail during midgestational development. Male mice are less affected by Smchd1-loss, with some (but not all) surviving to become fertile adults on the FVB/n genetic background. On other genetic backgrounds, all males lacking Smchd1 die perinatally. This suggests that, in addition to being critical for X inactivation, Smchd1 functions to control the expression of essential autosomal genes. RESULTS: Using genome-wide microarray expression profiling and RNA-seq, we have identified additional genes that fail X inactivation in female Smchd1 mutants and have identified autosomal genes in male mice where the normal expression pattern depends upon Smchd1. A subset of genes in the Snrpn imprinted gene cluster show an epigenetic signature and biallelic expression consistent with loss of imprinting in the absence of Smchd1. In addition, single nucleotide polymorphism analysis of expressed genes in the placenta shows that the Igf2r imprinted gene cluster is also disrupted, with Slc22a3 showing biallelic expression in the absence of Smchd1. In both cases, the disruption was not due to loss of the differential methylation that marks the imprint control region, but affected genes remote from this primary imprint controlling element. The clustered protocadherins (Pcdhα, Pcdhß, and Pcdhγ) also show altered expression levels, suggesting that their unique pattern of random combinatorial monoallelic expression might also be disrupted. CONCLUSIONS: Smchd1 has a role in the expression of several autosomal gene clusters that are subject to monoallelic expression, rather than being restricted to functioning uniquely in X inactivation. Our findings, combined with the recent report implicating heterozygous mutations of SMCHD1 as a causal factor in the digenically inherited muscular weakness syndrome facioscapulohumeral muscular dystrophy-2, highlight the potential importance of Smchd1 in the etiology of diverse human diseases.

The A-repeat links ASF/SF2-dependent Xist RNA processing with random choice during X inactivation.

One X chromosome, selected at random, is silenced in each female mammalian cell. Xist encodes a noncoding RNA that influences the probability that the cis-linked X chromosome will be silenced. We found that the A-repeat, a highly conserved element within Xist, is required for the accumulation of spliced Xist RNA. In addition, the A-repeat is necessary for X-inactivation to occur randomly. In combination, our data suggest that normal Xist RNA processing is important in the regulation of random X-inactivation. We propose that modulation of Xist RNA processing may be part of the stochastic process that determines which X chromosome will be inactivated.

Melanocyte homeostasis in vivo tolerates Rb1 loss in a developmentally independent fashion.

There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT-Cre mediated loss of exon 19 from a floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi-transformed phenotype. In this study, we show that Rb1-null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre-mediated ablation of the exon occurs. Further, Rb1 loss has no serious long-term ramifications on melanocyte homeostasis in vivo, with Rb1-null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre-mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.

Dual loss of rb1 and Trp53 in the adrenal medulla leads to spontaneous pheochromocytoma.

Using a Cre/loxP system, we have determined the phenotypic consequences attributable to in vivo deletion of both Rb1 and Trp53 in the mouse adrenal medulla. The coablation of these two tumor suppressor genes during embryogenesis did not disrupt adrenal gland development but resulted in the neoplastic transformation of the neural crest-derived adrenal medulla, yielding pheochromocytomas (PCCs) that developed with complete penetrance and were inevitably bilateral. Despite their typically benign status, these PCCs had profound ramifications on mouse vitality, with effected mice having a median survival of only 121 days. Evaluation of these PCCs by both immunohistochemistry and electron microscopy revealed that most Rb1(-/-):Trp53(-/-) chromaffin cells possessed atypical chromagenic vesicles that did not seem capable of appropriately storing synthesized catecholamines. The structural remodeling of the heart in mice harboring Rb1(-/-):Trp53(-/-) PCCs suggests that the mortality of these mice may be attributable to the inappropriate release of catecholamines from the mutated adrenal chromaffin cells. On the basis of the collective data from Rb1 and Trp53 knockout mouse models, it seems that the conversion of Rb1 loss-driven adrenal medulla hyperplasia to PCC can be greatly enhanced by the compound loss of Trp53, whereas the loss of Trp53 alone is generally ineffectual on adrenal chromaffin cell homeostasis. Consequently, the Trp53 tumor suppressor gene is an efficient genetic modifier of Rb1 loss in the development of PCC, and their compound loss in the adrenal medulla has a profound impact on both cellular homeostasis and animal vitality.

The glycosylphosphatidylinositol-anchored serine protease PRSS21 (testisin) imparts murine epididymal sperm cell maturation and fertilizing ability.

An estimated 25%-40% of infertile men have idiopathic infertility associated with deficient sperm numbers and quality. Here, we identify the membrane-anchored serine protease PRSS21, also known as testisin, to be a novel proteolytic factor that directs epididymal sperm cell maturation and sperm-fertilizing ability. PRSS21-deficient spermatozoa show decreased motility, angulated and curled tails, fragile necks, and dramatically increased susceptibility to decapitation. These defects reflect aberrant maturation during passage through the epididymis, because histological and electron microscopic structural analyses showed an increased tendency for curled and detached tails as spermatozoa transit from the corpus to the cauda epididymis. Cauda epididymal spermatozoa deficient in PRSS21 fail to mount a swelling response when exposed to hypotonic conditions, suggesting an impaired ability to respond to osmotic challenges facing maturing spermatozoa in the female reproductive tract. These data suggest that aberrant regulation of PRSS21 may underlie certain secondary male infertility syndromes, such as "easily decapitated" spermatozoa in humans.

Tissue-specific ablation of Prkar1a causes schwannomas by suppressing neurofibromatosis protein production.

Signaling events leading to Schwann cell tumor initiation have been extensively characterized in the context of neurofibromatosis (NF). Similar tumors are also observed in patients with the endocrine neoplasia syndrome Carney complex, which results from inactivating mutations in PRKAR1A. Loss of PRKAR1A causes enhanced protein kinase A activity, although the pathways leading to tumorigenesis are not well characterized. Tissue-specific ablation of Prkar1a in neural crest precursor cells (TEC3KO mice) causes schwannomas with nearly 80% penetrance by 10 months. These heterogeneous neoplasms were clinically characterized as genetically engineered mouse schwannomas, grades II and III. At the molecular level, analysis of the tumors revealed almost complete loss of both NF proteins, despite the fact that transcript levels were increased, implying posttranscriptional regulation. Although Erk and Akt signaling are typically enhanced in NF-associated tumors, we observed no activation of either of these pathways in TEC3KO tumors. Furthermore, the small G proteins Ras, Rac1, and RhoA are all known to be involved with NF signaling. In TEC3KO tumors, all three molecules showed modest increases in total protein, but only Rac1 showed significant activation. These data suggest that dysregulated protein kinase A activation causes tumorigenesis through pathways that overlap but are distinct from those described in NF tumorigenesis.

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice.

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.

Pocket protein function in melanocyte homeostasis and neoplasia.

Melanoma is the most lethal of human skin cancers and its incidence is increasing worldwide [L.K. Dennis (1999). Arch. Dermatol. 135, 275; C. Garbe et al. (2000). Cancer 89, 1269]. Melanomas often metastasize early during the course of the disease and are then highly intractable to current therapeutic regimens [M.F. Demierre and G. Merlino (2004). Curr. Oncol. Rep. 6, 406]. Consequently, understanding the factors that maintain melanocyte homeostasis and prevent their neoplastic transformation into melanoma is of utmost interest from the perspective of therapeutic interdiction. This review will focus on the role of the pocket proteins (PPs), Rb1 (retinoblastoma protein), retinoblastoma-like 1 (Rbl1 also known as p107) and retinoblastoma-like 2 (Rbl2 also known as p130), in melanocyte homeostasis, with particular emphasis on their functions in the cell cycle and the DNA damage repair response. The potential mechanisms of PP deregulation in melanoma and the possibility of PP-independent pathways to melanoma development will also be considered. Finally, the role of the PP family in ultraviolet radiation (UVR)-induced melanoma and the precise contribution that each PP family member makes to melanocyte homeostasis will be discussed in the context of a number of genetically engineered mouse models.

Transgenic overexpression of vascular endothelial growth factor-B isoforms by endothelial cells potentiates postnatal vessel growth in vivo and in vitro.

Vascular endothelial growth factors (VEGFs) play significant roles in endothelial growth, survival, and function, and their potential use as therapeutic agents to promote the revascularization of ischemic tissues in being avidly explored. VEGF-A has received most attention, as it is a potent stimulator of vascular growth. Results in clinical trials of VEGF-A as a therapeutic agent have fallen short of high expectations because of serious edematous side effects caused by its activity in promoting vascular permeability. VEGF-B, a related factor, binds some of the VEGF-A receptors but not to VEGF receptor 2, which is implicated in the vascular permeability promoting activity of VEGF-A. Despite little in vitro evidence to date for the ability of Vegf-B to directly promote angiogenesis, recent data indicate that it may promote postnatal vascular growth in mice, suggesting that it may have potential therapeutic application. We have specifically studied the effects of VEGF-B on vascular growth in vivo and on angiogenesis in vitro by analyzing transgenic mice in which individual isoforms (VEGFB167Tg and VEGFB186Tg) of VEGF-B are overexpressed in endothelial cells. VEGFB167Tg and VEGFB186Tg mice displayed enhanced vascular growth in the Matrigel assay in vivo and during cutaneous wound healing. In the aortic explant assay, explants from VEGFB167Tg and VEGFB186Tg mice displayed elevated vascular growth, suggesting a direct effect of VEGF-B isoforms in potentiating angiogenesis. These data support the use of VEGF-B as a therapeutic agent to promote vascular growth, in part, by potentiating angiogenesis. Furthermore, the lack of vascular permeability activity associated with either transgenic overexpression of the VEGF-B gene in endothelial cells or application of VEGF-B protein to the skin of mice in the Miles assay indicates that use of VEGF-B as a therapy should not be associated with edematous side effects.

Melanocytes in conditional Rb-/- mice are normal in vivo but exhibit proliferation and pigmentation defects in vitro.

The function of the retinoblastoma tumour suppressor (Rb1), and the pocket protein family in general, has been implicated as an important focal point for deregulation in many of the molecular pathways mutated in melanoma. We have focused on the role of Rb1 in mouse melanocyte homeostasis using gene targeting and Cre/loxP mediated tissue-specific deletion. We show that constitutive Cre-mediated ablation of Rb1 exon 2 prevents the production of Rb1 and recapitulates the phenotype encountered in other Rb1 knockout mouse models. Mice with conditional melanocyte-specific ablation of Rb1 manifest overtly normal pigmentation and are bereft of melanocytic hyperproliferative defects or apoptosis-induced depigmentation. Histologically, these mice have melanocyte morphology and distribution comparable with control littermates. In contrast, Rb1-null melanocytes removed from their in vivo micro-environment and cultured in vitro display some of the characteristics associated with a transformed phenotype. They proliferate at a heightened rate when compared with control melanocytes and have a decreased requirement for mitogens. With progressive culture the cells depigment at relatively early passage and display a gross morphology which, whilst reminiscent of early passage melanocytes, is generally different to equivalent passage control cells. These results indicate that Rb1 is dispensable for in vivo melanocyte homeostasis when its ablation is targeted from the melanoblast stage onwards, however, when cultured in vitro, Rb1 loss increases melanocyte growth but the cells are not fully transformed.

Inactivation of the murine Transferrin Receptor 2 gene using the Cre recombinase: loxP system.

Transferrin Receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 lead to type 3 hemochromatosis in humans. We have developed mice with a targeted deletion of TfR2. The Cre-recombinase:loxP system used to create the mice allows both full deletion and tissue-specific deletion of TfR2. The development of these mice will provide new models for type 3 hemochromatosis and assist in determining the role of TfR2 in iron metabolism.

Tyrosinase-Cre mice for tissue-specific gene ablation in neural crest and neuroepithelial-derived tissues.

This study describes the derivation of two new lines of transgenic mice that express Cre recombinase under the control of tyrosinase transcriptional elements. To determine the suitability of the Tyrosinase-Cre transgene for tissue-specific gene ablation studies, a fate map of Cre expression domains was determined using the Z/AP reporter strain. It was shown that Cre-expressing cells contribute to a wide array of neural crest and neuroepithelial-derived lineages. The melanocytes of the harderian gland and eye choroid, sympathetic cephalic ganglia, leptomeninges of the telencephalon, as well as cranial nerves (V), (VII), and (IX) are derived either fully or partly from Cre-expressing cephalic crest. The cells contributing to the cranial nerves were the first to exhibit Cre expression at E10.5 as they were migrating into the branchial arches. The melanocytes, chromaffin cells of the adrenal medulla, and dorsal root ganglia are derived from trunk neural crest that either express Cre or were derived from Cre-expressing precursors. An array of brain tissue including the basal forebrain, hippocampus, olfactory bulb, and the granule cell layer of the lateral cerebellum, as well as the retinal pigmented epithelium and glia of the optic nerve originate from Cre-expressing neuroepithelial cells.

Vegfb gene knockout mice display reduced pathology and synovial angiogenesis in both antigen-induced and collagen-induced models of arthritis.

OBJECTIVE: To determine the role of vascular endothelial growth factor B (VEGF-B) in 2 mouse models of arthritis, antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA). METHODS: For AIA studies, monarticular AIA was induced by methylated bovine serum albumin (mBSA) priming of Vegfb gene knockout (Vegfb(-/-)) and wild-type (Vegfb(+/+)) mice, followed by intraarticular injection of mBSA or saline control 8 days later. CIA was induced in Vegfb(-/-) and Vegfb(+/+) mice by intradermal injection of chick type II collagen in adjuvant. Arthritis was monitored in both models using defined criteria (clinical and histologic). Angiogenesis was measured by synovial vessel density in diseased and control joints. RESULTS: In AIA studies, Vegfb(+/+) mice displayed significant knee joint swelling and synovial inflammation 7 days after intraarticular injection of antigen. Synovial inflammation was associated with angiogenesis, since vessel density in AIA synovium was significantly higher in arthritic than in control joints from the same animal. Knee joint swelling, synovial inflammation, and inflammation-associated vessel density in arthritic joints were reduced in Vegfb(-/-) mice compared with arthritic joints from Vegfb(+/+) mice. Similarly, in CIA, both disease incidence and mean clinical severity scores were significantly reduced in Vegfb(-/-) mice compared with Vegfb(+/+) mice. Mean histologic severity scores and mean synovial vessel density were reduced in diseased joints from Vegfb(-/-) mice when compared with diseased joints from Vegfb(+/+) mice. CONCLUSION: The reduction in inflammation-associated synovial angiogenesis in Vegfb(-/-) mice implicates VEGF-B in pathologic vascular remodeling in inflammatory arthritis. VEGF-B may be an attractive target in the design of anti-angiogenic therapies for rheumatoid arthritis.